Kudo Yuma, Suzuki Hiroyuki, Tanaka Tomohiro, Kaneko Mika K, Kato Yukinari
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan.
Antibodies (Basel). 2023 Apr 28;12(2):31. doi: 10.3390/antib12020031.
Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant's (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (CMab-3; IgG, kappa) recognized peptides of the variant-5-encoded region, indicating that CMab-3 is a specific mAb for CD44v5. Moreover, CMab-3 reacted with CHO/CD44v3-10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent of CMab-3 for CHO/CD44v3-10 and PK-1 was 1.3 × 10 M and 2.6 × 10 M, respectively. CMab-3 could detect the exogenous CD44v3-10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that CMab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.
由于缺乏早期诊断生物标志物以及对传统化疗的耐药性，胰腺癌的预后较差。CD44已被公认为癌症干细胞标志物，并在多种癌症中发挥肿瘤促进和耐药作用。特别是，剪接变体在许多癌症中过度表达，并在癌症干性、侵袭或转移以及对治疗的耐药性中起重要作用。因此，了解每个CD44变体（CD44v）在癌症中的功能和分布对于建立以CD44为靶点的肿瘤治疗至关重要。在本研究中，我们用CD44v3 - 10过表达的中国仓鼠卵巢（CHO）- K1细胞免疫小鼠，并建立了多种抗CD44单克隆抗体（mAb）。其中一个建立的克隆（CMab - 3；IgG，κ）识别变体5编码区域的肽段，表明CMab - 3是CD44v5的特异性单克隆抗体。此外，通过流式细胞术，CMab - 3与CHO/CD44v3 - 10细胞或胰腺癌细胞系（PK - 1和PK - 8）发生反应。CMab - 3对CHO/CD44v3 - 10和PK - 1的表观亲和力分别为1.3×10⁻⁸M和2.6×10⁻⁸M。在蛋白质印迹中，CMab - 3可以检测外源性CD44v3 - 10和内源性CD44v5，并且在免疫组织化学中，它能对福尔马林固定石蜡包埋的胰腺癌细胞进行染色，但对正常胰腺上皮细胞不染色。这些结果表明，CMab - 3在各种应用中可用于检测CD44v5，有望用于胰腺癌的诊断和治疗应用。
